Immune maturation is viewed as a Darwinian process that involves continuous selection of the structure (s) of best fit. The long term objective of this study is to understand the molecular basis of antigen recognition and its contribution as a selective force in shaping the antibody response to phosphocholine-protein (PC- KLH) conjugates. The evolution of antibodies (in particular Group II) recognizing different molecular aspects of the PCcarrier complex will be studied using a combination of molecular genetic and physical measurements of serum antibodies, hybridomas and monoclonal antibodies obtained at various times during the immune response to PC-KLH. The specific aims are 1) Determine the molecular progression of the antibody response to PC-KLH via hybridoma analysis. The emergence of Group II antibodies will be determined and analyzed for isotype and/or clonotype restriction. The role of somatic mutation will be determined and specific gene usage will be assessed. 2) Determine the vulnerability of the T15+ clonotype to antigen-binding loss mutations. 3) Analyze the evolution of epitope recognition with regard to fine specificity, affinity, and physical interaction between structural features of phenyl-PC and the active site. 4) Analyze the relative contributions of PC-phenyl-tyrosine and PC-phenyl-histidine to evolution of the Group II response. The methods to be used include mRNA sequencing, in situ hybridoma lysis and mRNA hybridization to obtain gene usage information. Affinity of monoclonal antibodies will be measured by fluorescence spectroscopy. Antibody fine specificity will be assessed using a panel of hapten analogs. Physical interactions between antibody combining sites and structural components of the hapten will be assessed by nuclear magnetic resonance spectroscopy.